In day-to-day dynamic activities where sensory input is abundant, stimulus representations in the visual cortex are modulated based on their attentional priority. Several studies have established the top-down role of a fronto-parietal dorsal attention network in selective attention. In the current study, we aimed to investigate whether activity of subregions of this network and the visual cortex is modulated by feature-based attentional weighting, and if so, whether their timecourses of activity are correlated. To this end, we analyzed fMRI data of 28 healthy subjects, who performed a feature-based go/no-go task. Participants had to attend to one or two colored streams of sinusoidal gratings and respond to each grating in the task-relevant stream(s) except to a single non-target grating. Univariate and multivariate fMRI results indicated that activity in bilateral fronto-parietal  and visual  regions was modulated by selecting one instead of attending to two gratings. Functional connectivity was not significantly different between fronto-parietal and visual regions when attending to one as opposed to two gratings. Our study demonstrates that activity in subregions of both the fronto-parietal and visual cortex is modified by feature-based attentional weighting. Since the limited capacity of the brain impedes us to process all sensory stimuli at once, selective attention is a crucial ability in daily life . DependiIn order to resolve the competition of sensory input for processing resources, it has been established that selective attention modifies stimulus representations in the visual cortex based on behavioral relevance . As a reSeveral studies have investigated the origin of top-down signals to the visual cortex to gain insight in the process of selective attention. The fronto-parietal dorsal attention network (DAN), encompassing the frontal eye fields (FEF), intraparietal sulcus (IPS) and superior parietal lobe (SPL), has been reported to play a key role in each mechanism of selective attention: spatial-based , object-In the current study, we aimed to investigate whether activity in subregions of the DAN and the visual cortex is modulated by feature-based attentional weighting, and if so, if the respective timecourses of activity suggest an interaction between fronto-parietal and visual regions. To this end, participants performed a go/no-go task under fMRI, in which two differentially colored streams of stimuli were displayed. Here, different task conditions required participants to either select one stream or attend to both streams. Task conditions were sensorially matched and only differed in the need to prioritize information based on one or two colors. The task of the current study is different from those in previous feature-based attention studies e.g.,  in that Twenty-eight healthy volunteers  participated in the current study. All participants had normal or corrected-to-normal vision and all of them reported not to suffer from color blindness. The study was approved by the Ethics Committee Research UZ/KU Leuven (Reference number: S60929). All participants gave written informed consent in accordance with the Declaration of Helsinki.Stimulus presentation and response registration were controlled by a personal computer running Presentation 19.0 . Participants performed a feature-based go/no-go task in the MR scanner . The tasThe feature-based go/no-go task had a mixed experimental design, with each participant completing nine fMRI runs of nine experimental blocks each. Experimental blocks began by presenting a cue for 2,400 ms, indicating the task-relevant streams in the upcoming block, followed by a jittered fixation interval between 2 and 4 s. The cue was a fixation dot that doubled in size and had one out of three possible colors: cyan or magenta (one of both streams task-relevant), or yellow (both streams task-relevant). Each sequence of three blocks contained each of these conditions exactly once in a fixed order within runs, with participants being informed about their order prior to each run. In experimental blocks where one stream of gratings was task-relevant, participants were instructed to attend to the cued (= task-relevant) stream and ignore the uncued (= task-irrelevant) stream. In experimental blocks where both streams of gratings were task-relevant, the two streams had to be attended simultaneously. The order of the blocks was counterbalanced across runs and participants. After each block, a fixation period of 6 s occurred.Each experimental block consisted of 18 task trials, resulting in a total of 162 trials per run. Gratings had nine possible orientations ranging from 10° to 170° with a 20° interval. In each sequence of nine trials, each stream contained each of nine possible orientations exactly once in random order, with the restriction that co-presented gratings were never the same. Participants were instructed to fixate the center of the screen and press a button on the response box with their right thumb every time a target (grating ≠ 90°) was present in the task-relevant stream(s) . However, participants had to inhibit the response when a non-target (grating = 90°) was present in a task-relevant stream . Participants were asked to prioritize accuracy over speed while performing the task. The number of no-go trials was matched for blocks where either one or two streams of gratings required attention by presenting a grating with a random target orientation instead of the grating with the non-target orientation for each sequence of nine trials of one stream in blocks where both streams were task-relevant.Stimulus presentation and response registration were controlled by a personal computer running the Psychophysics Toolbox 3.0.11 in MATLAB . Following a standard procedure from 1-weighted three-dimensional turbo-field-echo sequence . For task and meridian mapping runs, respectively, 209 and 251 whole-brain functional MRI volumes were acquired using a single-shot echo-planar imaging sequence .Structural and functional magnetic resonance images were acquired through a 3T Philips Ingenia CX scanner with a 32-channel head coil at the Department of Radiology of the University Hospitals Leuven. A high-resolution structural scan was obtained through a Ta priori power calculation  with results of our previous study   and two us study . Behavious study  and the us study . fMRI daN = 11) were preprocessed by excluding task trials in which 30% or more of the samples were not successfully recorded during stimulus presentation. Data analysis consisted of calculating the proportion of trials for each task condition (attend to one or two streams of gratings) and each participant in which at least one saccade to at least one of the two stimuli occurred. Here, only eye movements with a minimal duration of 10 ms and an amplitude that fell within the respective x- and y-boundaries of the stimuli counted as saccades. Additionally, for the trials in which at least one saccade occurred, the average number of saccades per trial was calculated. Paired t-tests (α = 0.05) were performed to allow the pairwise comparison of the aforementioned variables between task conditions.Eye tracking data were collected for fixation control during task runs using the Eyelink 1000 eye tracking system . For the eye tracking data analyses, 11 out of 24 participants were excluded due to software issues that impeded the task and eye tracking data to be synchronized during the data acquisition. Additionally, 2 out of 24 participants were excluded based on eye tracking data not being successfully obtained in more than 2/3 task runs. The eye tracking data of all included participants  for correct go trials. Trials with anticipatory RTs of 100 ms or less were excluded from the behavioral analysis. Accuracy levels on go and no-go trials were analyzed on a trial-by-trial level by applying a Poisson mixed-effects model with a random slope. On the other hand, RTs on correct go trials were analyzed on a trial-by-trial level by using a linear mixed-effects model with a random slope. Both the results on accuracy and RTs were presented as averaged values per participant.1-weighted) image and spatial normalization into Montreal Neurological Institute (MNI) space while re-sampling to a voxel size of 3 mm × 3 mm × 3 mm (= 27 mm3). The functional images of task runs used in the univariate whole-brain analysis, as well as those of meridian mapping runs, were additionally preprocessed by performing spatial smoothing with an 8 mm full-width half-maximum kernel.The preprocessing stream for functional images of the feature-based go/no-go task used in the univariate region of interest (ROI) analysis was the same as for those used in the multivariate analysis. The first two functional images of each run in each participant were excluded from preprocessing and subsequent statistical analyses . The preThe preprocessing stream for functional images of the feature-based go/no-go task in the functional connectivity analysis included spatial normalization into MNI space, scrubbing, high-pass filtering above 0.01 Hz, regressing out white matter and ventricle signals, regressing out head motion, regressing out task-evoked activity using regressors of each of the respective conditions and low-pass filtering below 0.10 Hz. Here, the regressors of the task conditions were included to filter out statistical associations between timeseries of ROIs related to the timing of the task .Multiple ROIs were defined for the univariate, multivariate and functional connectivity analyses . Fronto-t-test. These first-level contrasts were combined in a group-level statistical analysis by estimating a random-effects model. Next, beta weights were extracted from the random-effects model for the bilateral fronto-parietal  and visual  ROIs using the MarsBaR toolbox (t-test (α = 0.05) to compare the beta weights between task conditions. On the other hand, in the univariate whole-brain analysis, first-level contrasts between the task conditions were defined by using a paired t-test to determine whether activity in a respective voxel was stronger in one of both conditions, followed by a group-level statistical analysis with a random-effects model. The result of the univariate whole-brain analysis was presented with bspmview1 as a statistical parametric map relying on cluster-extent based thresholding, with the primary voxel-level threshold set to p < 0.001 and the secondary cluster-level threshold to p < 0.05 (corrected for multiple comparisons with the family wise error (FWE) method)  with the duration set from cue onset to mask offset in the last trial of the block. Additionally, the GLM contained six motion regressors, one regressor for each translation and rotation in the three dimensions of space. After estimating the parameters of the GLM, in the univariate ROI analysis, first-level contrasts were defined for each condition vs. baseline by using a paired  toolbox , followe method) .To investigate whether activity levels in these block-level univariate analyses are biased by the cues, no-go trials and/or trials with mistakes, the result of the block-level whole-brain analysis was compared with that of an event-related  whole-brain analysis in which the correct go trials of each task condition were contrasted. This analysis was performed in the same way as the block-level whole-brain analysis, except that the GLM had five task regressors: two for the correct go trials of each condition (attend to one or two streams of gratings), one for all cues, one for all correct no-go trials and one for all trials with mistakes.t-statistics per run for the condition in which two streams were attended, the first-level contrast of the respective condition vs. baseline was calculated in the GLM. On the other hand, to obtain the t-statistics per run for the condition in which one stream was attended, the average first-level contrast of the magenta and cyan subconditions vs. baseline was calculated in the GLM. In this way, an equal amount of response patterns was obtained for the conditions (attend to one or two streams of gratings), which were used as input for a linear support vector machine (SVM). The cross-validation with the SVM was performed by training the classifier on a subset of response patterns of task runs (∼70%) to find the hyperplane that best separates the conditions where either one or two gratings were attended in each participant, followed by testing the classifier on the remaining task runs (∼30%). This k-fold cross-validation procedure (with the exact amount of folds depending on the number of included runs for the respective participant) was performed for several iterations until each possible combination of training and test set was acquired. The multivariate analysis resulted in one decoding accuracy averaged across folds and iterations for each participant and each ROI, reflecting the specificity of response patterns  for different distributions of attention. Decoding accuracies were compared with chance-level performance  by using a paired t-test (α = 0.05).The multivariate analysis was performed using CoSMoMVPA  and LIBSt-test on the Z-values of individual participants. In addition, we obtained the contrast between conditions where either one or two gratings were attended by running a paired t-test on their respective Z-values. For each contrast, the significance level of α = 0.05 was corrected for multiple comparisons  with the false discovery rate (FDR) method.To investigate differences in the task-related interaction of fronto-parietal and visual ROIs between task conditions (attend to one or two streams of gratings), a functional connectivity analysis was performed using custom-made scripts written in MATLAB . Similart(10) = −1.64, p = 0.13] (t(10) = −0.53, p = 0.61] ,  = 0.13] . The ave = 0.61] .Z = 0.33, p = 0.74]  = 6.49, p = 1.38 × 10–6] ,  = 0.74] . Similar = 0.41] . On the  × 10–6] .Beta weights (averaged across voxels) were significantly higher when selecting one stream instead of attending to two streams of gratings for the ROI corresponding to the bilateral SPL . On the Congruent with the ROI analysis, in the (block-level) whole-brain analysis, attending to one stream instead of two streams of gratings resulted in higher activity in the bilateral SPL (extending into the precuneus and posterior cingulate cortex) and lower activity in the right IPS and bilateral occipital/occipitotemporal cortex . AdditioResults of the trial-by-trial whole-brain analysis on the correct go trials were congruent with those of the block-level whole-brain analysis . Additiot(23) = 7.05, p = 3.49 × 10–7; IPS: Δ = 0.19, 95% CI , t(23) = 8.86, p = 7.20 × 10–9; SPL: Δ = 0.12, 95% CI , t(23) = 6.35, p = 1.75 × 10–6] (t(23) = 2.84, p = 9.24 × 10–3; V2: Δ = 0.07, 95% CI , t(23) = 5.28, p = 2.35 × 10–5; V3: Δ = 0.06, 95% CI , t(23) = 3.45, p = 2.16 × 10–3; V4: Δ = 0.03, 95% CI , t(23) = 1.68, p = 0.11; LOC: Δ = 0.07, 95% CI , t(23) = 5.34, p = 2.00 × 10–5; FG: Δ = 0.08, 95% CI , t(23) = 5.78, p = 6.91 × 10–6] ,  × 10–6] . Similar × 10–6] .t(23) = 7.75, p = 7.33 × 10–8; IPS: Δ = 0.14, 95% CI , t(23) = 9.28, p = 3.08 × 10–9; SPL: Δ = 0.11, 95% CI , t(23) = 6.34, p = 1.79 × 10–6; V1: Δ = 0.04, 95% CI , t(23) = 3.47, p = 2.08 × 10–3; V2: Δ = 0.04, 95% CI , t(23) = 2.55, p = 0.02; V3: Δ = 0.04, 95% CI , t(23) = 3.19, p = 4.06 × 10–3; V4: Δ = 0.02, 95% CI , t(23) = 1.60, p = 0.12; LOC: Δ = 0.04, 95% CI , t(23) = 3.12, p = 4.76 × 10–3; FG: Δ = 0.04, 95% CI , t(23) = 2.93, p = 7.47 × 10–3].Considering the differences in the number of voxels between ROIs , the mult(23) = 1.67, p = 0.05] and the bilateral SPL and LOC .Selecting one stream compared to attending to two streams of gratings did not result in any significant differences in functional connectivity between ROIs , althougThe current study aimed to investigate whether activity in subregions of the DAN and the visual cortex is modulated by feature-based attentional weighting, and if so, whether their timecourses are correlated. Results of both the univariate ROI and whole-brain analysis showed that activity levels for subregions of the DAN and visual cortex were modulated by attending to one as opposed to two streams of gratings, with higher activity levels in the SPL and lower activity levels in the IPS and occipital/occipitotemporal regions , 3 acrosN = 100) did not lead to substantially different findings. Thus, attention-related effects in activity patterns of the color-sensitive region V4 were most likely too small to be detected by the classifier when dissociating between conditions in which feature values were identical.Prioritizing one or two streams of gratings based on color led to different activity levels in several regions in the visual cortex. In the univariate ROI analysis, bilateral visual regions  had decreased activity when attending to one instead of two streams of gratings. This is congruent with results of the univariate whole-brain analysis, where a decrease in activity for bilateral visual regions (mostly corresponding to FG) was observed. Since the stimulus display was sensorially matched between task conditions, these results can be attributed to a top-down modulation of stimulus representations in the visual cortex e.g.,  and are Noteworthy, congruent with results of the univariate analyses, decoding accuracies in the multivariate analysis were most reliable on the level of high-order visual regions. Several neuroimaging studies have showed that different perceptual representations of objects  modulate activity levels in the high-order visual cortex . TherefoNot only activity in visual regions, but also activity in regions of the DAN was modulated by the distribution of attention. The multivariate analysis showed that activity patterns in all subregions of the DAN  are modulated by feature-based attentional weighting. Congruently, the univariate ROI and whole-brain analysis demonstrated differences in activity levels for regions of the DAN between task conditions. Specifically, bilateral SPL activity was higher when attending to one instead of two streams of gratings. This could be related to the involvement of the SPL in shifting attention , where tA possible confound for results on the level of fronto-parietal regions that needs to be addressed is a difference in difficulty level between task conditions. Considering that task difficulty modulates activity in fronto-parietal dorsal attention regions e.g.,  and sincThe functional connectivity analysis revealed that functional connectivity was not significantly different between fronto-parietal and visual regions when attending to one as opposed to two streams of gratings. A trend toward significantly increased functional connectivity between bilateral IPS and LOC, as well as between bilateral SPL and LOC, can be observed that is unlikely to be related to object perception, because the involvement of parietal regions in perceptual processes is related to global rather than local processing e.g., . TherefoWhile most evidence on a top-down role of the DAN in selective attention centers around its parietal subregions, several studies argue that top-down signals originate in the FEF . Here, sThe go/no-go task used in the current study relied on feature-based attentional weighting, where the features  of the stimuli determined their behavioral relevance. However, while feature-based attentional weighting was required to identify the relevant stimulus, this process was most likely followed by spatial mechanisms to shift attention toward and to enhance stimulus processing at its respective location. It is important to note that the spatial component is much smaller than the feature component in the task of the current study since the stimuli were presented in close proximity at the fovea. Furthermore, an interaction of spatial- and feature-based attention regularly occurs in real-life situations and is inherent to the way in which visual attention is organized, that is through topographic maps of the environment which represent the attentional priority of items in the visual field based on their bottom-up and top-down values . Thus, eThe current study demonstrates that activity in all regions of the DAN, as well as activity in low- to high-order visual regions, is modified by feature-based attentional weighting. Further research is required to elucidate whether subregions of the DAN, either directly or indirectly, have a top-down role in modifying stimulus representations in the visual cortex based on feature priority.celine.gillebert@kuleuven.be.The datasets presented in this article are not readily available because the ethics approval does not permit the public archiving of data supporting the current study, and sharing of data requires a formal data-sharing agreement in accordance with ethical procedures governing the re-use of sensitive data. Requests to access the datasets should be directed to the corresponding author CG: The studies involving human participants were reviewed and approved by Ethics Committee Research UZ/KU Leuven. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.AL was responsible for the data collection and for drafting the manuscript. All authors contributed to the design and data analysis of the current study, as well as to the revision the manuscript, and approved the final version of the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aims to establish and validate a predictive model based on radiomics features, clinical features, and radiation therapy (RT) dosimetric parameters for overall survival (OS) in hepatocellular carcinoma (HCC) patients treated with RT for portal vein tumor thrombosis (PVTT).n = 105) and validation (n = 26) cohorts. The clinical target volume was contoured on pre-RT computed tomography images and 48 textural features were extracted. The least absolute shrinkage and selection operator regression was used to determine the radiomics score (rad-score). A nomogram based on rad-score, clinical features, and dosimetric parameters was developed using the results of multivariate regression analysis. The predictive nomogram was evaluated using Harrell’s concordance index (C-index), area under the curve (AUC), and calibration curve.We retrospectively reviewed 131 patients. Patients were randomly divided into the training  and an AUC of 0.71 . The predictive accuracy was assessed by a calibration curve.The radiomics-based predictive model significantly improved OS prediction in HCC patients treated with RT for PVTT. Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer death worldwide. The prognosis of HCC is poor, with a 5-year survival rate of 5%–18% . ApproxiHCC can be diagnosed on contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) . Therefon = 105) and validation cohort (n = 26), with a ratio of 4:1.We retrospectively reviewed HCC patients newly diagnosed with PVTT between December 2007 and December 2019 in one institution. A contrast-enhanced CT or MRI was performed for diagnosis and staging. According to the 7th edition American Joint Committee on Cancer/American Joint Committee on Cancer staging system, all patients were staged IIIB . All patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2. In this study, patients were either inoperable or not eligible for TACE or TARE. The primary treatments were RT and/or targeted therapy. Patients with a history of OP, RT, TACE, or TARE were excluded. A total of 131 patients were enrolled and randomly divided into the training cohort  was delineated using the diagnostic and simulation images of the PVTT with or without the primary liver tumor. The CTV was determined by expanding the GTV margin by 5–10 mm to consider areas at significant risk of microscopic disease. The planning target volume (PTV) was generated by adding a 5–10–mm margin to the CTV in all directions for a setup error. RT was delivered using either three-dimensional conformal radiotherapy or intensity-modulated radiation therapy (IMRT) based on physician preference. The treatment plans were designed using 6- or 10-MV photons. All patients were treated with linear accelerators. Dosimetric parameters such as the dose of the CTV and normal organs were extracted from RT planning systems . The prescribed dose was 45, 50, or 60 Gy delivered in 1.8–2 Gy per fraction (BED10: 53.1–72.0 Gy). The goals were to deliver the prescribed dose to ≥95% of the PTV and 95% of the prescribed dose to ≥99% of the PTV. The dosimetric parameters were recorded for evaluation. After RT, abdominal CT or MRI was performed for response assessment. Most patients underwent abdominal CT or MRI 1 month after RT. The patency status of the portal vein area was evaluated by experienced radiologists.Contrast-enhanced CT was performed using Philips MRC 800  with a peak tube voltage of 120 kVp, tube current of 325 mA, rotation time of 0.75 s, matrix of 512 × 512, field of view of 50 cm, and slice thickness of 3 mm for RT planning and radiomics analysis.http://www.lifexsoft.org; Orsay, France) , was contoured by experienced radiation oncologists on all axial CT images. Segmentation was performed using the Eclipse system. Three-dimension ROI was visualized using Local Image Features Extraction (LIFEx) version 5.10 ( France) . The LIF France) Figure 1https://www.r-project.org; Vienna, Austria) to make randomization. The least absolute shrinkage and selection operator (LASSO) Cox regression was performed to determine the radiomics features that can predict OS in the training cohort. We performed the 10-fold cross-validation 20 times. The final value of lambda  was determined with the minimized mean deviance and the corresponding subset of covariates with non-zero coefficients. Features were selected by the total times of non-zero coefficient in 20 randomized 10-fold cross-validations. The Cox proportional-hazard model was fitted with the selected features, and the radiomics score (rad-score) predicting OS could be calculated linearly.The study population was divided into the training and validation cohorts in a ratio of 4:1 using the sample function of R (version 3.6.1) software , white blood cell, platelet, albumin, alanine aminotransferase, aspartate aminotransferase (AST), total bilirubin, creatinine, and prothrombin time.The prescribed RT doses, RT fields as involved PVTT with or without primary liver tumors, CTV, normal liver volume (NLV), and mean liver doses (MLDs) of all patients were recorded Figure 2U test was used to assess continuous variables. Univariate Cox regression analysis was performed to determine the predictors of OS from rad-score, clinical features, and RT dosimetric parameters. Thereafter, multivariate Cox regression analysis was used to select prognostic factors for the establishment of predictive nomogram models. The “survival” and “rms” packages were used for survival analysis, nomogram model construction, Harrell’s concordance index (C-index) calculation, and calibration curve. The C-index was a measure of goodness-of-fit for outcomes in a regression model, ranging from 0.5 to 1. A C-index value of 0.5 indicated that the predictive ability of the model was no better than a random chance, whereas C-index values of >0.7 and >0.8 indicated that the model was good and strong, respectively. A value of 1 implied that the model perfectly predicted the outcome. The “survivalROC” package was used for calculation and comparison of the area under the curve (AUC) of the receiver operating characteristic (ROC) curve for evaluation of the nomogram. The AUC ranged from 0.5 to 1. The discrimination potent of the model based on the value of AUC was as follows: 0.5, no discrimination potent; 0.7–0.8, acceptable; 0.8–0.9, excellent; and >0.9, outstanding. Differences were considered significant at p < 0.05.Statistical analysis was performed using R (version 3.6.1) and SPSS version 24.0 . Numerical data are presented as mean ± standard deviation. LASSO regression analysis was performed using the “glmnet” package to select the radiomics features for rad-score to predict OS. The optimal cutoff value of the rad-score was determined using X-tile software . The survival curves were plotted using the Kaplan–Meier method and assessed using the log-rank test. The Chi-square test was used to assess categorical variables, and the Mann–Whitney Based on the results of multivariate Cox regression analysis, the nomogram models with significant clinical features, RT dosimetric parameters, and/or rad-score were constructed to predict OS in HCC patients treated with RT for PVTT. The confirmation of nomograms was subjected to a 1,000 resampling bootstrap analysis for validation. The predictive models were evaluated with C-indexes, AUC of ROC curves, and calibration curves Figure 1This study was approved by the Institutional Review Board of our institution (IRB number: 20MMHIS215e).A summary of the baseline characteristics of the 131 patients is presented in 3 . The NLV was 1,140.7 ± 480.8 cm3, and the MLD was 1891.3 ± 651.5 cGy. The RT dosimetric parameters for the training and validation cohorts are summarized in The RT dosimetric parameters for 131 patients are presented in The treatment outcomes of the patients are presented in A total of 48 radiomics features were extracted from the imaging data of all patients. Two features were selected by LASSO Cox regression analysis to predict the OS in the training cohort Figure 3p = 0.007). Considering the training cohort, the median OS rates in the high- and low-risk groups were 7.5 months  and 11.8 months , respectively (p = 0.038). In the validation cohort, the median OS rates in the high- and low-risk groups were 6.8 months  and 12.6 months , respectively (p = 0.033). The median OS rates were significantly lower in the high-risk groups than in the low-risk groups in both the training and validation cohorts  and low-risk (<−0.6) groups based on the cutoff value of the rad-score. The median OS rates in the high- and low-risk groups were 7.4 months  and 12.4 months , respectively  and American College of Radiology (ACR) guidelines  and standard quality assurance measures.Few studies have reported the application of radiomics and clinical features to predict treatment outcomes and prognosis in different types of cancers treated with RT. Hou et al. established an integrated model that combined posttreatment CT radiomics features and clinical features for response and OS prediction in esophageal cancer patients undergoing neoadjuvant chemoradiotherapy . Wu et aThis study has several limitations. First, MRI is the preferred imaging modality for the evaluation of liver lesions. The technique of MRI-guided RT with MRI simulation and planning is rapidly developing . HoweverRadiomics features combined with clinical features and dosimetric parameters have better performance than each significant feature and clinical nomogram. This study recommends the development of a predictive model with significant clinical features, radiomics features, and dosimetric parameters. The multicenter analysis is warranted after the standardization of treatment protocol, radiology imaging, and radiomics data in all hospitals for external validation to ensure the accuracy of the universal predictive model.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by MacKay Memorial Hospital. The patients/participants provided their written informed consent to participate in this study.Conceptualization: Y-MH and Y-JC. Methodology: Y-MH, H-CT, S-MH, and Y-JC. Software: Y-MH Validation: Y-MH, S-MH, and Y-JC. Formal analysis: Y-MH. Investigation: Y-MH. Resources: Y-MH, T-EW, M-JC, C-CL, C-WC, and Y-JC. Data curation: Y-MH. Writing—original draft preparation: Y-MH. Writing—review and editing: Y-MH, S-MH, and Y-JC. Visualization: Y-MH. Supervision: Y-JC. Project administration: Y-JC. Funding acquisition: Y-JC. All authors have read and agreed to the published version of the manuscript.This research was funded by MacKay Memorial Hospital (Grant numbers MMH-E-109-13 and MMH-E-110-13) and the Ministry of Science and Technology of Taiwan (Grant number MOST 109-2314-B-195-003-MY3).We would like to thank Editage for providing their editing services.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Head and neck squamous cell carcinoma (HNSCC) arises from the mucosal epithelium in the oral cavity, pharynx, sino-nasal region, and larynx. Laryngeal squamous cell carcinoma (LSCC) represents one-third of all head and neck cancers. Dysregulated RNA-related pathways define an important molecular signature in this aggressive carcinoma. The Survival Motor Neuron (SMN) protein regulates fundamental aspects of the RNA metabolism but, curiously, its role in cancer is virtually unknown. For the first time, here, we focus on the SMN in the cancer context. We conducted a pilot study in a total of 20 patients with LSCC where the SMN was found overexpressed at both the protein and transcript levels. By a cellular model of human laryngeal carcinoma, we demonstrated that the SMN impacts cancer-relevant behaviors and perturbs key players of cell migration, invasion, and adhesion. Furthermore, in LSCC we showed a physical interaction between the SMN and the epidermal growth factor receptor (EGFR), whose overexpression is an important feature in these tumors. This study proposes the SMN protein as a novel therapeutic target in LSSC and likely in the whole spectrum of HNSCC. Overall, we provide the first analysis of the SMN in human cancer. Head and neck squamous cell carcinomas (HNSCCs) represent a heterogeneous group of tumors that arise from the mucosal epithelium of the oral cavity, pharynx, nasal cavity and paranasal sinuses, and larynx . They reHere, we explored the expression levels of SMNs in laryngeal squamous cell carcinoma. We first performed a pilot study in a cohort of 20 patients with LSCC. We showed that the SMN is upregulated in LSCC tissues at both the transcript and protein levels. In HLaC-79 cells, a human LSCC cell line, the SMN impacts cancer-relevant behaviors, such as cell proliferation, cell migration, and cisplatin sensitivity. Furthermore, in agreement with our previous study , SMN-defOur intent was to explore a relationship between the RNA-binding protein SMN and LSCC. To this end, we conducted a pilot study in a cohort of 20 treatment-naive patients with LSCC see A. It is Collectively, by this pilot study we provide evidence that the SMN is overexpressed in LSCC.To gain insights into the role of the SMN in LSCC, we conducted in vitro studies using HLaC-79 cells, a LSCC cell line ,34. For It has been established that β-actin plays an essential role in regulating cell migration ,36. NotaIt is known that reduced E-cadherin expression allows the conversion of static and polarized epithelial cells into dynamic and invasive cells . NotablyEGFR overexpression occurs with high frequency in HNSCC . IdentifAltogether, this pioneering study suggests that the RNA-binding protein SMN may be an intriguing player in LSCC and likely in the whole spectrum of head and neck tumors.RNA-binding proteins (RBPs) help to maintain cell homeostasis, tuning regulatory networks implicated in processes such as proliferation, differentiation, and metabolism . Based oSMN influences the RNA life cycle at multiple levels, and it enables cells to finely regulate gene expression in time and space . Due to RNA-related pathways seem to act as driving forces for HNSCC development . Basic aHere, we provide for the first time a demonstration that SMN is overexpressed in LSCC, which represents one-fourth of all head and neck cancers . By a piFurthermore, a potential role of the SMN in HNSCC was corroborated by in vitro studies using a cellular model of human LSCC. We demonstrated that SMN is required to sustain cancer-relevant behaviors, such as cell proliferation and migratory capability. We also observed that SMN knockdown increases cisplatin sensitivity. This is a critical issue in the HNSCC context. Cisplatin is still the anticancer drug for advanced HNSCC. However, HNSCCs exhibit different levels of cisplatin resistance. Cisplatin administration in resistant patients could generate almost no beneficial effect but could increase the chance of adverse side effects and tumor progression. At this stage, we can only speculate that molecular strategies targeting SMN in HNSCC could modulate the resistance to cisplatin-based chemotherapy. To gain insights into cellular activities concerning the actin cytoskeleton, cell–cell contact, and cell migration, we explored the effects of SMN knockdown on E-cadherin and β-actin proteins. By an evaluation of E-cadherin expression levels in SMN knockdown cells, we provided an important novelty in the context of both the SMN and HNSCC. Our findings suggest that the SMN could be involved in mechanisms regulating E-cadherin expression. Noteworthy, this result agrees with our Western blot analysis, revealing that SMN expression levels are inversely correlated with E-cadherin changes in LSCC cells. This strongly suggests an implication of the SMN in the regulatory networks underlying cell adhesion and extracellular matrix platforms. We think that an interplay between the SMN and the E-cadherin pathway may be crucial, since a loss of E-cadherin has been reported to trigger epithelial–mesenchymal transition in several cancers, including HNSCC ,46. FutuOverall, to our knowledge this is the first study focusing on the RNA-binding protein SMN in cancer. Here, we provide evidence that the SMN could play a pivotal role in HNSCC biology. Although the underlying molecular mechanisms need further characterizations, the SMN emerges as a new attractive therapeutic target in LSSC and likely in the whole spectrum of HNSCC.The following antibodies were used: anti-SMN mouse monoclonal antibody ; anti-SMN rabbit polyclonal antibody ; anti-SMN rabbit monoclonal antibody ; anti-GAPDH mouse monoclonal antibody ; EGFR rabbit polyclonal antibody ; anti-Ki67 mouse monoclonal antibody ; anti-RPS6 mouse monoclonal antibody ; anti-E-cadherin mouse monoclonal antibody ; anti-vimentin mouse monoclonal antibody ; and anti-vimentin rabbit monoclonal antibody . The secondary antibodies conjugated to horseradish peroxidase were purchased from Jackson ImmunoResearch Laboratories, Suffolk, UK, and used at a dilution of 1:5000. The Alexa Fluor488- and the Alexa Fluor594-conjugated secondary antibodies were purchased from Thermo Fisher Scientific Inc., Waltham, MA, USA, and were used at a dilution of 1:250. The Alexa-Fluor-594-conjugated phalloidin were from Life Technologies, Carlsbad, CA, USA. MTT, 3--2,5-diphenyltetrazolium bromide and cisplatin, cis-Diammineplatinum(II) dichloride were purchased from Sigma-Aldrich, Co., St. Louis, MO, USA.The list of oligos used in this study is indicated in A total of 20 patients with LSCC were included in this study. The sites of the tumors and staging and grading according to the American Joint Committee on Cancer  are summ2 humidified atmosphere, at 37 °C. For knockdown experiments, cells were transfected with 3 unique 27mer siRNA duplexes targeting the human SMN1 gene or with trilencer-27 scrambled negative control . Lipofectamine 2000 (Life Technologies) was used as the transfection reagent, according to the manufacturer’s instructions. Cells were harvested after 48 or 72 h post transfection.A HLaC-79 laryngeal carcinoma cell line, kindly supplied from Marianne Schmidt, Klinik und Poliklinik für Hals-, Nasen- und Ohrenkranke Labor Josef-Schneider-Str. 11 97,080 Würzburg, was cultured in a RPMI 1640 medium  supplemented with heat-inactivated 10% FBS (Gibco), penicillin-streptomycin (Gibco), and GlutaMAX (Gibco), in a 5% CO3 cell/well) in a 96-well plate were transfected with siControl or siSMN siRNA and grown in a complete culture medium for 24 h. Next, cells were treated or not with 10 μg/mL cisplatin for a further 24 h. After incubation, MTT -2,5-diphenyltetrazolium bromide) was added to each well at a final concentration of 0.5 mg/mL. Following 3 h of incubation at 37 °C, dimethyl sulfoxide was added to dissolve the crystals. The absorbance was determined at 577 using a spectrophotometer microplate reader .Cell viability and proliferation were assessed by an MTT test. HLaC-79 cells , at 0 and 20 h. Images were captured by a SPOT RT3 camera and elaborated by IAS 2000 v.5.0.1 software . The healing rate was quantified by measuring the distance between cells at the edges of the wound by using ImageJ, National Institutes of Health, USA 1.53a software.Formalin-fixed paraffin-embedded (FFPE) samples from two patients  to reduce nonspecific background. After two washes in PBS, slides were processed for the padlock assay as previously described . When thHLaC-79 cells and four μm thick sections obtained from the FFPE blocks were subjected to in situ PLA using Duolink In Situ Detection Reagents Green and Orange kit , according to the manufacturer’s instructions. A combination of primary antibodies to the SMN  and EGFR  were used. The PLA signal was detected by a epi-fluorescence microscope .g at 4 °C. The protein concentration was measured by a QubitTM fluorometer , according to the manufacturer’s instructions. Protein extracts were stored at −80 °C until use.A protein extraction was obtained from frozen tissues processed in a lysis buffer  supplemented with a complete protease and phosphatase inhibitor cocktail , using a homogenizer . The homogenates were boiled for 10 min and centrifugated for 20 min at 12,000× g for 10 min at 4 °C. The protein concentration was measured by a QubitTM fluorometer (Invitrogen), according to the manufacturer’s instructions. Protein extracts were stored at −80 °C until use.HLaC-79 cells were processed in a lysis buffer  supplemented with a complete protease and phosphatase inhibitor cocktail . Extracts were passed five times through a 25 G needle, incubated on ice for 15 min, and clarified at 12,000× Protein extracts were electrophoresed through standard 10% SDS-PAGE or NuPAGE 4–12% (Life Technologies Corporation) and transferred onto nitrocellulose membranes . The immunodetection of the reactive bands was revealed by chemiluminescence  and analyzed by an iBright 1500 (Thermo Fisher Scientific Inc.).Cellular extracts were prepared in an IP Buffer  and a complete protease and phosphatase inhibitor cocktail . Immunoprecipitation assays were performed overnight at 4 °C following the standard procedure, using the anti-EGFR rabbit polyclonal antibody. As a negative control, the immunoprecipitation was carried out with rabbit IgG beads (Thermo Fisher Scientific Inc.). After five washes in an IP buffer, the immunoprecipitated complexes were eluted by boiling in a Laemmly’s buffer for 10 min and analyzed by SDS-PAGE on 10% polyacrylamide gel followed by immunoblotting.® reagent according to the manufacturer’s instructions. The RNA from the HLaC-79 cells was then reverse transcribed using a High-Capacity cDNA Reverse Transcription kit . A semiquantitative PCR (RT-PCR) assay was performed in triplicate using the BioMix 2X  according to the manufacturer’s instructions. The RNA from the frozen tissues was treated to remove residual amounts of genomic DNA by DNase treatment, according to the following protocol: 4 μg of RNA were incubated with 1.4 units of DNase I  at 37 °C for 10 min. The sample was then treated with 1× EDTA  at 75 °C for 10 min, to deactivate the enzyme. Afterwards, 1 μg of DNase-treated RNA was reverse transcribed using the iScript cDNA Synthesis kit , containing both oligo-dT and random primers, according to the manufacturer’s protocol.The total RNA from the cells and frozen tissues was extracted using TRIzolDroplet Digital PCR was performed using a 1× QX200 EvaGreen ddPCR Supermix (Bio-Rad), 100 nM of each primer, and 0.5 µL of each cDNA sample, according to the supplier’s specifications. Water-in-oil droplets were generated from the sample using the QX200 Droplet Generator (Bio-Rad) with its microfluidics system. The PCR was successively carried out using a C1000 Touch thermal cycler (Bio-Rad) following the Bio-Rad standard amplification protocol. Finally, the droplets underwent the QX200 Droplet Reader (Bio-Rad) and QuantaSoft software version 1.7.4 (Bio-Rad) was used to analyze the results.t-test or one-way or two-way ANOVA test with a Bonferroni test for multiple comparison as specified in the figure legends; p < 0.01 was considered statistically significant.All experiments were performed on at least three independent biological replicates. Data are presented as mean ± S.D. Statistical analysis was performed using the GraphPad Prism 9.4.1 software. Data were analyzed using an unpaired


Background: About 90% of liver cancer-related deaths are caused by hepatocellular carcinoma (HCC). N7-methylguanosine (m7G) modification is associated with the biological process and regulation of various diseases. To the best of our knowledge, its role in the pathogenesis and prognosis of HCC has not been thoroughly investigated.Aim: To identify N7-methylguanosine (m7G) related prognostic biomarkers in HCC. Furthermore, we also studied the association of m7G–related prognostic gene signature with immune infiltration in HCC.Methods: The TCGA datasets were used as a training and GEO dataset “GSE76427” for validation of the results. Statistical analyses were performed using the R statistical software version 4.1.2.Results: Functional enrichment analysis identified some pathogenesis related to HCC. We identified 3 m7G-related genes  as prognostic biomarkers for HCC. A risk score was calculated from these 3 prognostic m7G-related genes which showed the high-risk group had a significantly poorer prognosis than the low-risk group in both training and validation datasets. The 3- and 5-years overall survival was predicted better with the risk score than the ideal model in the entire cohort in the predictive nomogram. Furthermore, immune checkpoint genes like CTLA4, HAVCR2, LAG3, and TIGT were expressed significantly higher in the high-risk group and the chemotherapy sensitivity analysis showed that the high-risk groups were responsive to sorafenib treatment.Conclusion: These 3 m7G genes related signature model can be used as prognostic biomarkers in HCC and a guide for immunotherapy and chemotherapy response. Future clinical study on this biomarker model is required to verify its clinical implications. Liver Cancer is the major cause of cancer-related mortality. Hepatocellular carcinoma (HCC) accounts for approximately 90% of liver cancer-related deaths . DespiteN7-methylguanosine (m7G), methylation on the N7 position of guanosine is a type of positively charged RNA modification in tRNA, rRNA, or internal mRNA regions . PreviouHerein, we perform a bioinformatics analysis to identify the association of several m7G-related genes in the molecular pathogenesis of HCC. Furthermore, we aim to construct a prediction model and nomogram to predict prognosis in patients with HCC, its relation with immune infiltration, and chemotherapy sensitivity.xenabrowser.net)]. Furthermore, to increase the reliability of our study, we extracted the RNA sequencing data of HCC patients and normal tissues from a GEO dataset “GSE76427” and used it for validation of the results from the TCGA datasets.The RNA sequencing data of the HCC patients, normal tissues, and their corresponding clinical information were obtained from the TCGA database on 30 June 2021. The TCGA datasets were downloaded from the UCSC Xane website [UCSC Xena (Lists of the m7G-related genes were extracted from the prior studies . The expp < 0.05) associated with differentially expressed m7G-related genes.The “clusterprofile”, “enrichplot”, “ggplot2”, “GOplot”, and “library (ReactomePA)” packages in R were used to perform Gene Ontology (GO) and Reactome pathway analysis to identify the significant pathways  regression was used to incorporate the m7G-related genes with Risk score = (expression level of Gene1 ∗ β1 + expression level of Gene2 ∗ β2 +...+expression level of Genen ∗ βn).The TCGA HCC cohorts were then divided into high- and low-risk groups based on the median risk score and the corresponding Kaplan-Meier analyses were performed comparing these two subgroups. Finally, the AUC was calculated for 3- and 5-year survival in the ROC curves.The “survival” “survminer”, and “rms” packages in R were used for the development of the nomogram. Univariate and multivariate cox regression models were used to identify the independent factors affecting the prognosis of HCC patients. Based on the clinicopathologic features of m7G-related genes from multivariate analysis, a cox-proportional hazard (PH) model was used to develop a nomogram to predict the survival probability. The goodness-of-fit test purposed by Schoenfeld was used to test the validity of PH models.50) data matrix of the sorafenib with the “pRRophetic” package and analyzed the IC50 difference between the high-risk and low-risk groups.CYBERSORT tool was used to estimate the expression of 22 different immune cells in HCC patients. We calculated the immune, stromal, and ESTIMATE scores in the high and low-risk groups by using “estimate” package in R. Furthermore, the relation of risk-score with immune checkpoints and immune-suppressive cytokines was also assessed. Furthermore, we checked if the risk model could serve as a potential predictor of chemotherapeutic sensitivity to sorafenib. We converted the gene expression TCGA datasets into half inhibitory concentrations  with 95% confidence intervals (CI). Mann-Whitney test was used to compare the immune infiltration and pathway activation between the two groups.Statistical analyses were performed using the R statistical software version 4.1.2. Gene expression levels between the HCC tumor and normal tissue were performed with a single-factor analysis of variance. Categorical variables were compared by using the Pearson chi-square test. Kaplan-Meier analysis was used to compare the survival outcomes. The univariate and multivariate Cox regression analyses were used to evaluate the independent prognostic value of the identified risk model. All the survival outcomes were presented as A total of 503 M7G-related genes were extracted from the previous studies. Out of them, 492 genes were expressed in HCC and 18 m7G-related genes were differentially expressed (upregulated: 5 and downregulated: 13) in the HCC than in the normal tissues . SignifiGO enrichment analysis of the differentially expressed genes showed that the m7G-related genes were associated with several biological processes, cellular components, and molecular functions. The m7G-related genes were significantly associated with the rhythmic process, microvillus, apical part of the cell, mitochondrial matrix, basolateral plasma membrane, actin-based cell projection, basal plasma membrane, and basal part of the cell . The Reap < 0.05), TYMS , AGXT , ANO1 , IYD , and PDGFRA ) were significantly associated with the prognosis of HCC (The Risk score: Risk score = (0.251) ∗ CDK1 + (−0.122) ∗ ANO1 + (0.318) ∗ PDGFRA. Kaplan-Meier survival curve showed that the prognosis of patients in the high-risk group was significantly poorer than those in the low-risk group (p < 0.05)   with AUC < 0.05) . As the  < 0.05) . A Heatm < 0.05) .p values for risk score age, gender, N-stage, T-stage, M-stage, tumor grade, and risk-score were 0.3689, 0.0605, 0.0896, 0.6864, 0.4132, and 0.8801 respectively. The p-value for the global Schoenfeld test was 0.7701    with AUC< 0.001) .With the advances in high-throughput sequencing technology, more and more studies have published different kinds of RNA modification including the internal mRNA m7G methylation. In clinical research, several previous studies identified the role of N6-methyladenosine (m6A) remodeling in the molecular pathogenesis of several types of carcinogenesis and the role of m6A-related genes in the prognosis of several types of cancers . M6A remOur study constructed an m7G associated prognostic model and risk score for predicting the prognosis of HCC which will help in understanding its molecular pathogenesis and will further benefit in understanding the diagnosis, treatment, and prognosis. The results of our study identified that CDK1, ANO1, and PGDFRA-based prognostic models can accurately predict the prognosis in patients with HCC. Mutation in m7G methyltransferase is associated with several diseases like Down syndrome, primordial dwarfism, ovarian carcinogenesis, etc., . FurtherCyclin-dependent kinase 1 (CDK1) is highly expressed in HCC tissues and is associated with poor prognosis . It may Results of the functional enrichment analysis of the m7G-related genes with GO revealed that some of them were associated with the pathogenesis of HCC. Mitochondrial dysregulation is associated with the aggressiveness of HCC . Actin−mImmune infiltration is also associated with carcinogenesis and the prognosis of HCC . MoreoveSeveral previous studies have shown the reliability of using risk scores for the prediction of the prognosis of HCC. Our risk-score model showed that the patients with high-risk scores had significantly worse survival than those with low-risk scores. The risk score was positively correlated with other well-known prognostic factors like T-stage, N-stage, M-stage, and tumor grade. Furthermore, a quantitative nomogram was developed with the inclusion of risk score and several well-known existing prognostic factors of HCC, and its prognostic ability is better than other existing clinicopathologic factors. This further improved the performance and reliability of the risk score. Therefore, we believe that the proposed model can be used for the stratification of prognosis and identify the appropriate therapies. Lastly, the risk score was used to assess the prognosis in a GEO dataset, which showed that the risk score can accurately predict the prognosis. Therefore, we believe with further validation from future clinical trials, this could be one of the novel prognostic models with higher accuracy.in vivo and in vitro experiments were not performed to verify the results obtained from our study. Second, the results of this study were not validated in the resected specimens of HCC from our institution. Despite these limitations, we believe the results of our study will provide knowledge about the prognostic significance of m7G-related genes in HCC and will certainly help in guiding future clinical studies.There are certain limitations to our study. First, Our study identified that the 3 m7G-related signature model can be used as a prognostic biomarker in HCC. The functional enrichment analysis of the m7G-related genes showed some of the molecules associated with the pathogenesis and pathways for the progression of HCC. In addition, m7G-related genes were also positively correlated with immune cell infiltration. Future clinical study on this biomarker model is required to verify its clinical implications.


Limulus amebocyte lysate (LAL) reaction-based assay, the most commonly used endotoxin detection method, requires a skilled technician. In this study, to develop an easy-to-use and highly sensitive endotoxin sensor, we created an electrochemical endotoxin sensor by using an interdigitated array electrode (IDAE) device with advantages of amplifiable signals via redox cycling and portability. We added Boc-Leu-Gly-Arg-p-aminophenol (LGR-pAP) as an electrochemical substrate for an LAL reaction and detected p-aminophenol (pAP) released from LGR-pAP as a product of an endotoxin-induced LAL reaction via an IDAE device. The IDAE device showed a great redox cycling efficiency of 79.8%, and a 4.79-fold signal amplification rate. Then, we confirmed that pAP was detectable in the presence of LGR-pAP through chronoamperometry with the potential of the anode stepped from −0.3 to 0.5 V vs. Ag/AgCl while the cathode was biased at −0.3 V vs. Ag/AgCl. Then, we performed an endotoxin assay by using the IDAE device. Our endotoxin sensor detected as low as 0.7 and 1.0 endotoxin unit/L after the LAL reaction for 1 h and 45 min, respectively, and these data were within the cut-off value for ultrapure dialysis fluid. Therefore, our highly sensitive endotoxin sensor is useful for ensuring medical safety.The  Limulus amebocyte lysate (LAL) assay based on an endotoxin-induced enzymatic LAL cascade reaction is the most widely used endotoxin detection method [p-nitroaniline released from Boc-Leu-Gly-Arg-p-nitroaniline (LGR-pNA) as a chromogenic substrate of the clotting enzyme [An endotoxin is a component of the outer membrane of Gram-negative bacteria . Endotoxn method . In thisn method  or by men method . Additiog enzyme ,11. Howe−4 ng/mL using a metal–organic framework [p-aminophenol (LGR-pAP) was used as a substrate of the clotting enzyme, and an endotoxin assay was performed by amperometrically detecting p-aminophenol released from LGR-pAP on an electrochemical chip device after 1 h of the LAL reaction [To overcome this problem, we have developed an easy-to-use LAL-based endotoxin sensor by using an electrochemical chip device. A sensor using an electrochemical chip device enables user-friendly detection because it is small, portable, and convenient . Previouramework . Tian etramework . Xie et ramework . In our reaction . Nonethereaction . To imprreaction . Furtherreaction . Redox cp-quinoneimine (pQI) at the cathode  device. The IDAE device comprises two comb-shaped electrodes interdigitated alternately. Oxidized species at the anode of the comb-shaped electrode are reduced at the cathode of the comb-shaped electrode; thereafter, the reduced species are oxidized at the anode comb again. This cycling of redox reaction amplifies the electrochemical signal ,21. The  cathode .6 EU/L of endotoxin stock solution and stored at 4 °C. Before being used, the endotoxin stock solution was vigorously mixed by vortexing for 30 min in accordance with the USP-RSE protocol. LGR-pAP was dissolved in endotoxin-free water to obtain 10 mmol/L stock solution and stored at −20 °C.The following materials were used in this study: United States Pharmacopeia reference standard endotoxin (USP-RSE) and a LAL-based endotoxin assay kit Endospecy-24S set comprising assay buffer and LAL reagents ; an endotoxin-free water ; pAP and KCl ; LGR-pAP ; 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid ; and negative photoresists of ZPN1150 and SU-8 3005 . USP-RSE was diluted with endotoxin-free water to obtain 2 × 10An IDAE device was fabricated via a lift-off process involving standard photolithography and sputtering. The photoresist of ZPN1150 was patterned on a glass slide; then, Ti/Pt/Au was sputtered on the glass slide by using a sputtering system . After lift-off in acetone, the photoresist of SU-8 3005 was patterned on the glass slide to insulate the device, except the sensor area. The IDAE device consisted of two comb-shaped electrodes as working electrodes and an Au flat plate electrode as a counter electrode on the glass slide. According to the photomask designs, the width of each comb and the distance between the combs were 10 μm, and the non-insulated length of the finger of each comb was 1.0 mm.Electrochemical measurements were performed using a Ag/AgCl electrode (saturated KCl) as a reference electrode and an Au flat plate as a counter electrode in a multichannel potentiostat . A sample solution (300 μL) was introduced to the IDAE device, and the Ag/AgCl electrode was inserted into the solution. After one measurement, the IDAE device was washed with water for repeated use. Because the obtained currents from the IDAE device gradually decrease by electrode fouling with the repeated use of the device, we set the times for repeated used of an IDAE device to be up to three.An electrochemical endotoxin assay was performed in accordance with our previously described methods . The assIc/Ia) was 79.8%, which indicated that most of the oxidized FMA at the anode reached the cathode. The green line in Ia/Is), because of the signal amplification of redox cycling. Furthermore, the measured anodic current under the redox cycling condition was compared with the theoretical value by using Equation (1) [The IDAE device was fabricated and characterized. tion (1) :(1)Is=mbIs is the steady-state current under redox cycling with the IDAE device; m and b are the number and length of the fingers of the anode comb-shaped electrode, respectively; n is the number of electrons involved in a redox reaction; F is Faraday’s constant; c and D are the concentration and diffusion coefficient of the redox molecule, respectively; and w and wg are the total width of the fingers of the anode and cathode comb-shaped electrodes and the gap width between the anode and cathode, respectively. The theoretical steady-state current of the anode was 1.45 μA, where m = 40, b = 1.0 mm, n = 1, F = 9.65 × 104 C/mol, c = 500 μmol/L, and D = 7.6 × 10−10 m2/s according to a previous report [w and wg were 20 and 10 μm, respectively, which roughly matched the steady-state current of the anode at 0.6 V vs. Ag/AgCl in In Equation (1), s report ; w and wpAP was detected in the presence of LGR-pAP to characterize our assay system because the solution after the LAL reaction contained pAP and LGR-pAP. Cyclic voltammetry for 500 μmol/L pAP or LGR-pAP in 50 mmol/L HEPES buffer (pH 7.8) containing 0.1 mol/L KCl was performed using the IDAE device to determine the applied potential for the amperometric detection of pAP in the presence of LGR-pAP. The potential of the anode was swept at 50 mV/s, while the cathode was biased at −0.3 V vs. Ag/AgCl. The endotoxin assay was performed using the IDAE device. The LAL reaction with the endotoxin was induced using 0.5 mmol/L LGR-pAP for 1 h, 45 min, or 30 min at 37 °C. Thereafter, pAP released from LGR-pAP was detected using the IDAE device when the potential of the anode was stepped from −0.3 V to 0.5 V vs. Ag/AgCl, while the cathode was biased at −0.3 V vs. Ag/AgCl. In this study, a highly sensitive endotoxin sensor involving redox cycling was developed by using an IDAE device. The characterization of the fabricated IDAE device indicated that the IDAE device had a great redox cycling efficiency of 79.8% and 4.79-fold signal amplification rate. Cyclic voltammetry and chronoamperometry for pAP and LGR-pAP in HEPES buffer revealed that pAP was detectable in the presence of LGR-pAP, and the potential of the anode and cathode could be biased at 0.5 and −0.3 V vs. Ag/AgCl, respectively. The endotoxin assay demonstrated that values as low as 0.7, 1.0, and 100 EU/L endotoxin could be detected after the LAL reaction of 1 h, 45 min, and 30 min, respectively. The limit of detection of our sensor covered the cut-off value of ultrapure dialysis fluid (1.0 EU/L) with 1 h and 45 min of the LAL reaction time. In the future, the sensitivity of our sensor will be improved to reach 1.0 EU/L with 30 min of the LAL reaction time by using a LAL reagent without a chromogenic substrate and an IDEA device with reduced electrode distance for improved redox cycling efficiency. In the future, we will perform real sample analysis, selectivity characterization, and a comparison of analytical performance of our sensor with the conventional LAL-based endotoxin assay for the realization of practical use. Our highly sensitive endotoxin sensor will be used to ensure medical safety.


Mindfulness-based cognitive therapy (MBCT) is a promising alternative treatment for generalized anxiety disorder (GAD). The objective of this study was to examine whether the efficacy of group MBCT adapted for treating GAD (MBCT-A) was noninferior to group cognitive behavioural therapy (CBT) designed to treat GAD (CBT-A), which was considered one of first-line treatments for GAD patients. We also explored the efficacy of MBCT-A in symptomatic GAD patients compared with CBT-A for a variety of outcomes of anxiety symptoms, as well as depressive symptoms, overall illness severity, quality of life and mindfulness.n = 138) were randomized to MBCT-A or CBT-A in addition to treatment as usual (TAU). The primary outcome was the anxiety response rate assessed at 8 weeks after treatment as measured using the Hamilton Anxiety Scale (HAMA). Secondary outcomes included anxiety remission rates, scores on the HAMA, the state-trait anxiety inventory (STAI), the Hamilton Depression Scale (HAMD), the Severity Subscale of the Clinical Global Impression Scale (CGI-S), and the 12-item Short-Form Health Survey (SF-12), as well as mindfulness, which was measured by the Five Facet Mindfulness Questionnaire (FFMQ). Assessments were performed at baseline, 8 weeks after treatment, and 3 months after treatment. Both intention-to-treat (ITT) and per-protocol (PP) analyses were performed for primary analyses. The χ2 test and separate two-way mixed ANOVAs were used for the secondary analyses.This was a randomized, controlled, noninferiority trial with two arms involving symptomatic GAD patients. Adult patients with GAD ; PP rate difference = 5.85% ]. The anxiety remission rate, overall illness severity and mindfulness were significantly different between the two groups at 8 weeks. There were no significant differences between the two groups at the 3-month follow-up. No severe adverse events were identified.Our data indicate that MBCT-A was noninferior to CBT-A in reducing anxiety symptoms in GAD patients. Both interventions appeared to be effective for long-term benefits.ChiCTR1800019150, registration date: 27/10/2018).Registered at chictr.org.cn (registration number: The online version contains supplementary material available at 10.1186/s12888-022-04127-3. Generalized anxiety disorder (GAD) is one of the most prevalent psychiatric disorders and is characterized by chronic and persistent worry , 2. It iDue to the disadvantages of pharmacotherapy, including side effects, premature discontinuation, and a significant risk of relapse , 9, a laMindfulness is the awareness that emerges through intentionally focusing, in a nonjudgmental way, on how things are in the present moment . MindfulAlthough some randomized controlled trials (RCTs)  have evaOur primary objective was to compare MBCT-A with gold standard psychological treatment CBT-A to test the hypothesis that the anxiety response rate achieved with MBCT-A would not be more than 10% worse than that achieved with CBT-A. We hypothesized that MBCT-A would be noninferior to CBT-A in improving symptoms of anxiety. Our other objectives were to explore the effects of MBCT-A and CBT-A in terms of psychic and somatic anxiety symptoms, state and trait anxiety symptoms, depression symptoms, overall illness severity, quality of life and mindfulness.This study is a randomized, controlled, noninferiority trial with two groups: MBCT-A vs. CBT-A. Participants were recruited from the Outpatient Department of the Sixth Hospital of Peking University from November 2018 to November 2019 via (a) posters distributed in outpatient clinics and (b) recommendations from psychiatrists who worked in the Sixth Hospital of Peking University but were not involved in the study. All patients concurrently continued their regular outpatient psychiatry visits for medication management during the study period  at the Sixth Hospital of Peking University. The regular outpatient psychiatry visits did not include psychotherapy, and the average consulting time was approximately 10 min per patient, with an average of one visit every two weeks.All interested participants were assessed for eligibility. First, a trained research assistant screened all interested participants by telephone or in-person appointments using HAMA and a brief structured questionnaire. Additionally, the research assistant discussed the process of the study with interested participants. Next, for patients who passed the first screening, diagnostic screenings were independently performed by an attending psychiatrist in accordance with the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV). Finally, the principal investigator (B.X.H.) conducted a final screening using the study inclusion and exclusion criteria to determine eligibility. Informed consent forms were obtained from all eligible participants.We used the following inclusion criteria: (a) aged 18–65 years; (b) diagnosis of GAD based on DSM IV; (c) score ≥ 14 on the Hamilton Anxiety Rating Scale (HAMA) ; (d) medThe MBCT-A protocol largely followed the classical MBCT manual described by Segal, Williams, and Teasdale , 32. TheIn the MBCT-A manual, we made several adaptations to render the MBCT appropriate for treating GAD. First, the cognitive components of MBCT dealing with depression were replaced by components dealing with anxiety, which included psychoeducation about anxiety and automatic anxiety thoughts in session four, identifying warning signs for anxiety in session six. Second, the content of the precourse orientation was integrated into session one, a one-day retreat was shortened to 4 h and integrated into session six, and daily audio homework exercises were shortened to 30 min a day. Third, loving-kindness meditation was introduced in session two to cultivate kindness within GAD patients themselves and compassion for the somatic and psychic anxiety symptoms of GAD .PracticeQualified instructors with more than two years of experience delivered the MBCT-A program and were supervised by Dr. S. Helen Ma and Dr. Yen-Hui Lee, who both were Oxford mindfulness centres and approved the MBCT Teacher Trainer & Supervisor. The MBCT-A intervention consisted of weekly sessions for 8 weeks, each lasting 2 h involving up to 20–25 participants. All participants were instructed to report their daily mindfulness practice via a messaging and social media application (WeChat).The CBT-A group, as the active control intervention with the same protocol as in a previous trial , has beeTwo qualified therapists with either a psychiatry or psychotherapy background led each CBT-A group. The corresponding author X. B. H supervised the CBT-A therapists during the study period. Participants in the CBT-A group attended weekly 1.5 h sessions over 8 weeks, with 10–15 participants in each group. Each CBT-A session had a particular theme. Weekly homework was assigned at the end of each session and was handed in to the therapists and discussed in the following session. The session summary can be found in the Online Supplementary Protocol.The outcome measures were collected at baseline (T1), week 8 , and at a 3-month follow-up assessment (T3). Four trained psychiatric residents who were blinded to the patient’s treatment allocation conducted patient assessments. Demographic and baseline clinical information, including age, sex, education history, marital status, ethnicity, residential location, religious beliefs, age of onset, course of GAD, and use of medication, was collected using a questionnaire.The primary outcome was response rate: percentage of participants who received a ≥ 50% HAMA decrease relative to the baseline at 8 weeks. The secondary outcomes included the response rate at the 3-month follow-up, remission rate at the 8-week and 3-month follow-ups, and the symptoms of total, psychological, and somatic anxiety, state and trait anxiety, depression, overall illness severity, quality of life and mindfulness. Any severe adverse events were observed throughout the whole intervention process.The HAMA  is a 14-We calculated the sample size to investigate whether MBCT-A was not inferior to CBT-A. Because no previous studies directly compared MBCT with high-intensity CBT for the treatment of GAD, we used the findings from two studies – one that compared the effects of CBT and medication  and anotRandomization was performed using computer-generated random numbers that were generated by an independent statistician. A block randomization procedure (blocks of 6) with no stratification was used. Those involved in the trial were blinded to the block sizes.Sealed envelopes were used to conceal the randomization sequence. The intervention types were written on sheets of paper that were placed inside opaque envelopes. After the informed consent forms were signed, a trained research assistant opened the envelopes in order and noted the group assignment for the corresponding participants. The participants were informed of their group allocation immediately after randomization, and they were notified that the treatments received in both study groups could be helpful in improving anxiety symptoms. The participants who agreed to receive the allocation were informed to wait for notice of the group starting from the research assistant. The study flow diagram is shown in Fig. 1.The researchers were blinded to the randomization. The independent assessors were blinded to the treatment allocation. The analyst was blinded to the identity of the participants until the results were finalized.p < 0.05.Statistics were conducted using IBM SPSS Statistics ver. 22. The baseline characteristics of the two groups were compared using an independent samples t-test or Mann–Whitney’s U test for continuous variables and the χ2 test for categorical variables. For the primary outcome, both intention-to-treat (ITT) (including the participants who attended at least one session) and per-protocol (PP) analyses (including the participants who attended at least five out of eight sessions) were performed, and the 95% CI level was used to interpret the differences in MBCT-A and CBT-A response rates at 8 weeks. For the secondary analyses, only PP analyses were performed. We used the χ2 test to analyse the response rates at the 3-month follow-up and remission rates in the two groups at each assessment time . The effect size estimates were presented using Cohen’s d and were interpreted as small effects (0.2–0.5), moderate effects (0.5 to 0.8), and large effects (≥ 0.8) . We perfOut of the 682 screened participants, 168 (24.6%) were successfully recruited . A total of 138 participants finally attended the intervention sessions, including 82 women and 56 men, with a mean age of 35.94 (SD = 11.05) years. Of all the recruited participants, 17.9%  did not attend any intervention sessions. There was no difference in this proportion between the two groups. This was viewed as pretreatment attrition and was not included in the data analyses.n = 69) and CBT-A group (n = 69).The basic participant demographics and baseline clinical data for both groups are listed in Table p = 0.005) but no significant differences in other characteristics at baseline. The dropout rates in the MBCT-A and CBT-A groups were 15.9% and 18.8% at 8 weeks (p = 0.653) and 18.8% and 21.7% at the 3-month follow-up (p = 0.672), respectively.There were 58 84.1%) and 56 (81.2%) participants in the MBCT-A and CBT-A groups, respectively, who attended at least 5 out of the 8 sessions. The reasons for those dropouts included the following: more than 3 sessions absent without reason, time restriction , withdrawn consent without reason after the first session, and suspicious pulmonary tuberculosis after the third session  in ITT analysis and 5.85%  in PP analysis   nor the response rate  were significantly different between the two groups  = 0.649, P = 0.424. Additionally, the CGI-S scores revealed significant time simple effects between the 8-week assessment and the three-month follow-up assessment (p = 0.02). Both of these results indicate that the enhanced improvements in MBCT-A had not persisted at the three-month follow-up assessment.The CGI-S scores revealed significant group simple effects at the 8-week assessment, F = 11.403, P = 0.028. Thus, while the level of mindfulness increased in both groups immediately posttreatment, the increase was significantly greater in the MBCT-A group. There were no significant group simple effects in STAI-S and STAI-T scores at the 8-week assessment and no significant group simple effects in any STAI-S, STAI-T, CGI-S, or FFMQ scores at the 3-month follow-up assessments.Comparing the FFMQ scores revealed a significant simple effect of group at 8 weeks, F = 5.104, Mean values for all outcomes at all time points are reported for patients who completed the entire trial ; PP response rate difference = 5.85% ]. After treatment, 86.2% of the patients in the MBCT-A group and 80.4% of the patients in the CBT-A group achieved a response  and in HAMA remission rate (p = 0.994) at the 3-month follow-up visit . Second, there was a drop (63.8–48.2%) in the HAMA remission rate in the MBCT-A group but not the CBT-A group (44.6–48.1%) (shown in Table Second, the two groups had a different trend in HAMA remission rate during the treatment and follow-up. First, at the 8-week assessment, the MBCT-A group had a significantly higher remission rate compared with CBT-A group:63.8% in MBCT-A and 44.6% in CBT-A ( 63.8–48.% in the Third, unlike anxiety symptoms, the trait-related outcomes, including trait anxiety and trait mindfulness, showed further improvements at the 3-month follow-up visit compared with at 8 weeks, although this was not statistically significant (shown in Fig. Fourth, MBCT-A was effective in improving a wider range of outcomes, including well-being, overall illness severity, state and trait anxiety symptoms and depression symptoms. These findings are not only concordant with our hypothesis, but are also in line with previous studies demonstrating the effectiveness of MBCT in treating GAD , 29.Our study has several strengths. To our knowledge, this is the first noninferiority RCT study to compare MBCT and high-intensity CBT among Chinese GAD patients. Additionally, there was high homogeneity in the participant diagnoses, adding to the generalizability of the findings. Furthermore, we adapted MBCT for treating GAD and the interventions were delivered by trained and experienced instructors in accordance with standard protocols, thus demonstrating implementation of the treatment for future reference.Our study also has several limitations. First, we did not have a no-treatment control or a wait-list control. Current evidence suggests that GAD is a chronic condition that is unlikely to improve naturally over time . ConsideMBCT-A was noninferior to CBT-A in reducing anxiety symptoms in GAD patients. Both interventions appeared to be effective for long-term benefits. Taking into account the differences in treatment priorities and components between MBCT-A and CBT-A, our results suggest that both evidence-based interventions are optional treatments for patients with GAD.Additional file 1.Additional file 2.Additional file 3.


Nature Communications 10.1038/s41467-022-32847-3, published online 10 September 2022Correction to: The original version of this Article contained formatting errors in Table 1. The time interval was reported as 11–127 ka, this has been corrected to 117–127 ka. The column labels and data were incorrectly aligned. This has been corrected in both the PDF and HTML versions of the Article.


Heterogeneity is a major feature of Leber's hereditary optic neuropathy (LHON) and has a significant impact on the manifestation and diagnosis of the disease. This study explored whether multiple variations in mitochondrial genes were associated with the heterogeneity, mainly phenotypic heterogeneity. Ophthalmic examinations were conducted in two probands with LHON with G11778A and multiple mitochondrial DNA gene (mtDNA) variants. Skin fibroblast cell lines were generated from patients and age- and sex-matched controls. ROS levels, mitochondrial membrane potential, cell energy respiration, and metabolic functions were measured. Flow cytometry and cell viability tests were performed to evaluate the cell apoptosis levels and fate. We found that cells with more mtDNA variants had higher ROS levels, lower mitochondrial membrane potential, and weaker respiratory function. Flow cytometry and cell viability testing showed that multiple mtDNA variants are associated with different levels of cell viability and apoptosis. In conclusion, we found that skin-derived fibroblast cells from G11778A LHON patients could be used as models for LHON research. Multi-mtDNA variants contribute to mitochondrial function variety, which may be associated with heterogeneity in patients with LHON. MT-ND4 11778G>A, MT-ND6 14484T>C, and MT-ND1 3460G>A account for the majority of LHON patients  is the most common hereditary optic neuropathy with an average onset around age 20. Patients usually suffer from painless, subacute, or rapid vision loss in both eyes  mutations, nuclear gene mutations and epigenetic inheritance are closely related to the occurrence and development of LHON  was 28 years old and experienced progressive visual impairment without other symptoms at the age of 12 years . In 2019The other proband (I'-2) was 32 years old. No genetic information about their ancestry was available as the patient was adopted. The first visit to the Henan Eye Hospital was in 2019, when the patient complained of progressively loss of vision starting 1 year ago without other symptoms. BCVA was 0.1 in the left eye and 0.15 in the right eye. In the 2 years of follow up, the patient's vision had progressively decreased. In March, 2021, the vision reduced to FC/10 cm in both eyes. Fundus examination revealed pathological optic disks with incomplete boundaries . SS-OCT Considering these clinical symptoms, genetic test results and no history of other diseases, we diagnosed LHON in both patients. Both cases were of the same sex, comparable age, and similar baseline BCVA and duration of follow-up. However, the severity and progression of the disease varied significantly. The visual acuity of patient F1 III-4 was 0.15 when he first came to the hospital and there was no further decline after 2 years. This patient was diagnosed with mild LHON. The vision of patient F2 I'-2 dropped from 0.15 to FC/10 cm in 2 years. This patient was diagnosed with severe LHON. SS-OCT further demonstrated that III-4 had optic fiber atrophy only in the nasal and temporal regions, whereas I'-2 showed atrophy in four quadrants.MT-ND, MT-CO, MT-ATP, and MT-Cyb). These variants included missense and synonymy. In proband III-4, six missense variants were present in the coding regions of mtDNA: MT-ND2 4824A>G, MT-ATP6 8794C>T, MT-ATP6 8860A>G, MT-ND4 11778G>A, MT-Cyb14766C>T, and MT-Cyb15326A>G. Proband I'-2 had 11 mistranslation variants in mtDNA, almost twice as many as III-4. These variant loci were MT-ND2 4833A>G, MT-ATP6 8701A>G, MT-ATP6 8860A>G, MT-ND3 10398A>G, MT-ND4 11778G>A, MT-Cyb 14766C>T, MT-Cyb 15323G>A, MT-Cyb 15326A>G, MT-Cyb 15497G>A, MT-Cyb 15860A>G, and MT-Cyb 15968T>C. A phylogenetic analysis was performed to evaluate the conservation of these variants and sequences from other organisms such as mice, bovine, and Xenopus laevis. Among the six missense variants in patient III-4, two variants were conserved in mammals, except for G11778A. Four of the 11 variants in addition to G11778A were conservative in patient I'-2, particularly MT-Cyb 15497G>A . Fibroblast cells derived from the skin of subject without known mtDNA or nuclear gene mutations were used as controls (iFB-NC). The binocular visual acuity of control subject was 1.0 and no abnormality was found in visual field examination and OCT fundus imaging. First, ROS levels were detected in iFB cell lines. Fluorescence images showed that the ROS positive cells of iFB-LHON were much more than that of iFB-NC cells. Meanwhile, compared with iFB-III-4, iFB-I'-2 cells had higher ROS levels . MitochoElectron microscopy was used to test whether multiple variants in mtDNA are associated with changes in mitochondrial morphology. The number of mitochondrial cristae was reduced in iFB-LHON cells, particularly in the iFB-I'-2 cell line , indicat+, NADH, and ATP in mitochondria of these cell lines. NAD+ levels were lower in iFB-LHON cells than in iFB-NC cells, and iFB-I'-2 cells had the lowest levels of NAD+ . When the patients first visited the hospital, their clinical manifestations were similar. However, in a 2-year follow up, the phenotype of the patients varied. The visual acuity of patient III-4 was stable, while that of patients I'-2 declined progressively. Fundus photographs and SS-OCT results further confirmed diseases heterogeneity.The two probands included in the study, both male and of similar age, were diagnosed with LHON based on their clinical symptoms and gene sequencing results  and evaluated the respiratory function of the cells by measuring the OCR values. The results showed that the basic OCR value of the cells was significantly lower than that of the control cells. Among the two patients, patient I'-2 had a lower OCR value, indicating that the respiratory capacity was more damaged than that of the mild patient. ATP-linked respiration reflects the ATP synthesis ability of cells, and also showed the lowest value in patient I'-2. This trend corresponded to the ATP levels in iFB cells. Subsequently, the maximum respiratory rate was generated by CCCP stimulation. Here, the OCR value showed a significant difference, indicating insufficiency of the patients' respiratory potential and abnormal respiratory chain function. The ROS level in the iFB-I'-2 cells was found to be higher, and the mitochondrial membrane potential decreased more significantly. Mitochondrial damage in patient I'-2 was severe.The mutant genes in the two patients were mainly concentrated in the coding region of In addition to their involvement in energy synthesis, mitochondria also play an important regulatory role in cell survival and apoptosis. The CCK8 test results showed that iFB-LHON and iFB-NC cells had similar viability and proliferation rates. However, when cells were treated with CCCP to simulate harmful external stimuli, the patients' iFB cells were more vulnerable to injury and iFB-I'-2 cells were the most affected. The results indicated that although mitochondrial dysfunction had little effect on the survival and proliferation of patient fibroblasts under normal conditions, iFB-LHON cells were more susceptible to stress. It is possible that the responses to stimuli in cells bearing more mtDNA variants were exaggerated.Due to the inability to obtain retinal tissue from patients, skin fibroblasts have recently been used as models for molecular studies of genetic eye diseases , color vision, visual field, slit-lamp biomicroscopy, intraocular pressure (IOP), and SS-OCT.Genomic DNA was isolated from peripheral blood of all subjects with the TIANGEN Blood DNA Kit . Nanodrop 2000 was used to detect the DNA concentrations. Concentrations above 25 μg/μl and OD260/280 between 1.8 and 2.0 were used for the next steps. Sequencing and alignment were performed on the Nextseq 500  platform. Filter the original read to delete low quality reads . Clean data were mapped using BWA (Burrows-Wheeler Alignment tool). The mtDNA was sequenced with a depth greater than X5000. The Sanger sequencing was conducted to verify disease-relevant mutations.2 dermal punch biopsies. Cells were transfected with the SV40 virus to prepare iFB cells. iFB cells were cultured in a complete fibroblast medium . The cells were generated with informed consent and ethics approval [HNEECKY-2019(12)].Human fibroblast cells were isolated from 2 mmFibroblasts were seeded in a 6-well plate and cultured overnight. DCFH-DA  was diluted in serum-free medium at a ratio of 1:1000. Diluted DCFH-DA was added to the wells and cultured in a 37°C incubator for 40 min. Fluorescent images were captured using a microscope after washing the cells thrice with serum-free medium.The mitochondrial membrane potential assay was performed using a JC-1 detection kit . The cells were incubated overnight in a 6-well plate. After washing the cells once with PBS, 1 ml JC-1 staining working solution and 1 ml serum-containing medium were added to each well. Cells were cultured at 37°C in an incubator for 20 min. After incubation, the supernatant was discarded and the cells were washed twice with JC-1 buffer. Finally, 2 ml of serum-containing medium was added to the cell wells, and green and red fluorescent photos were taken. The intensity ratio of green and red fluorescence represents the mitochondrial membrane potential.2 for 1 h. Four inclusions from the XFe cell Mito stress test kit  were diluted into the following concentrations and injected into the probe plate during detection: oligomycin (15 μM), p-trifluoromethoxy phenylhydrazone [20 μM], and a mixture of antimycin A (5 μM) and rotenone (5 μM). Cell numbers were calculated for normalization. The software Wave 2.6.3 was used for the analysis.The function of the respiratory chain is reflected in the oxygen consumption rate  of cells. The OCR values were measured using an XFe analyzer . Fibroblast cells were seeded in the seahorse plate  at 12,000 cells per well and incubated overnight. The cell medium was replaced with an XF base medium  containing 2 mM glutamine, 1 mM pyruvate, and 10 mM glucose; and the seahorse plate was placed in a 37°C incubator without COThe cells were cultured overnight in 96-well plate. The CCK8  was diluted with basal medium without serum at a ratio of 1:100 to prepare the CCK8 working solution. The CCK8 working solution (100 μl/well) was added and the cells were incubated for 3 h. The absorbance was then determined at 450 nm. The value at 0 h was taken as the origin to calculate the relative value at each time point.5 cells were collected after treatment with or without CCCP [40 μM]  for 6 h. Cells were washed using cold PBS and resuspended in 100 μl 1× binding buffer. PE-annexin V (5 μl) and 7-AAD (5 μl) were added to the tube and samples were incubated for 15 min at room temperature in the dark after gentle vortexing. Samples were added to 400 μl 1× binding buffer for flow cytometry.The apoptosis assay was performed following the instructions of the PE Annexin V Apoptosis Detection Kit I . Briefly, the number of 1 × 10+ were detected using an ATP assay kit  and a NAD+ assay kit  according to the manufacturer's instructions, and the protein concentration was used as criterion for normalization.ATP and NADCells were lysed in RIPA lysis buffer  with 1X protease Inhibitor cocktail  and incubated on ice for 30 min. After centrifugation at 12,000 rpm for 30 min, the supernatant was mixed using 5× loading buffer. Subsequently, the supernatants were boiled at 95°C for 5 min. The samples were electrophoresed using 12% SDS-PAGE  and transfected into PVDF membranes . After blocking with 5% milk powder  for 1 h, the membranes were incubated with primary antibodies overnight at 4°C: MT-ND1, MT-ND4, MT-CO IV , VDAC2 , cleaved-CASP3  and β-actin . After washing thrice, the membranes were incubated with secondary antibodies, goat anti-mouse IgG-HRP , and goat anti-rabbit IgG-HRP  for 1.5 h at room temperature. Finally, the membranes were exposed using a chemiluminescence apparatus .post hoc test was used to evaluate the differences. The statistical significance was set at p ≤ 0.05.Image Pro Plus  was used to analyze fluorescence intensity. GraphPad Prism 8.0  was used for statistical analyses. Data in the graphs are shown as means ± standard deviations. One-way analysis of variance (ANOVA) followed by the Dunnett's In conclusion, we found that in fibroblast cells derived from LHON patients baring G11778A mutation, multi-mtDNA variants might contribute to the variety of mitochondrial function and response to external stimulus. Thus, the multi-mtDNA variants may contribute to the heterogeneity of LHON.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of Henan Eye Hospital [HNEECKY-2019(12)]. The patients/participants provided their written informed consent to participate in this study.SY conducted experiments, data analysis, manuscript writing, and financial support. QZ, XJ, and LY conducted data analysis. YL and QG collected clinical data. MY constructed the fibroblast cells. FQ performed flow cytometry. BL conceptualized the design, provided the materials and the financial support, revised, and gave final approval for the manuscript. All authors contributed to the article and approved the submitted version.This research was funded by the Key Technologies Research and Development Program of Henan Science and Technology Bureau (212102311009), Natural Science Foundation of Henan Province (222300420195), and National Natural Science Foundation of China grants (82004001 and 82071008).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.